Journal Article

Single-molecule FRET imaging of GPCR dimers in living cells

Holsey, Michael D, Gilmore, Grant T, Zhou, Zhou, Kaliszewski, Megan J, Govindaraju Alekhya, Harikumar, Kaleeckal G, Miller, Laurence J, Pati, Avik K, McCauley, Mitchell D, Smith, Adam W, Blanchard, Scott C, Javitch, Jonathan A, Asher, Wesley B, Jaqaman Khuloud, Mathiasen Signe, Geggier, Peter, Meszaros Jozsef, Terry, Daniel S

2021

Overview

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.A suite of generally applicable methods and tools, developed to enable single-molecule FRET-based studies of transmembrane proteins diffusing in the cell membrane of living cells, was used to study the oligomerization and dynamics of GPCRs.

• Publisher: Nature Publishing Group
• Subject: Automation / Biophysics / Cell membranes / Community colleges / Dimers / Dynamic structural analysis / Energy transfer / Fluorescence / Fluorescence resonance energy transfer / G protein-coupled receptors / Glutamate receptors / Glutamic acid receptors (metabotropic) / Labeling / Mammalian cells / Membrane proteins / Monomers / Oligomerization / Physiology / Plasma / Proteins / Receptor density / Receptor mechanisms / Receptors / Surgeons